The present invention relates to a method of lysing a microorganism which makes it possible, in general, to disrupt the latter, especially the membrane of one or more cells, in order to release at least one nucleic material of interest, for example deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) to be treated subsequently, in particular to be analysed.
The document U.S. Pat. No. 5,643,767 discloses a method of lysis for separating the DNAs or RNAs of cells in a liquid solution. A container is used for this purpose which contains a solvent for extracting the RNA or DNA, and a plurality of particles having a diameter of 0.1 to 1 mm, and at least one larger particle having a diameter of 3 to 5 mm. A movement is applied to the container thus filled, namely a shaking movement. It is expressly indicated in this document that this movement is preferred to a rotating movement, as generated with a mixer or another homogenizer, because with a rotating movement the cells simply turn in the same direction, and do not effectively collide with each other and with the beads so as to be crushed between the beads.
The document U.S. Pat. No. 4,295,613 discloses an apparatus for disrupting bacteria, according to which the sample containing the cells is placed in a tube in contact with Line beads. The beads, having a diameter of the order of 70 to 110 xcexcm, are introduced into the tube, with the sample to be treated and any required buffer solution, and then an oscillating movement is applied to the container or tube along a horizontal axis, in order to obtain the subcellular constituents in solution such as enzymes, proteins, carbohydrates and the like.
The document FR-A-1,576,299 discloses a method for disrupting plant and animal cells, according to which a particulate material is used whose particle size is from 0.05 to 1 mm in diameter, it being possible for the particles to be made of steel or another similar material. A movement is applied to the mixture of biological sample and particles, according to a laminar flow.
The document EP-A-0,317,803 discloses a method for generating multilamellar liposomes encapsulating an aqueous medium. The method consists in mixing lipids and the aqueous medium to be encapsulated, in stirring the mixture in a container in the presence of particles having a diameter of less than 3 mm, the preferred size being 50 to 100 xcexcm, in order to obtain liposomes having a diameter of about 150 to 3000 nanometers.
The document EP-A-0,796,917 discloses a device which releases particles into a biological sample containing cells. When the sample is stirred or sonicated, this device comprises a partition which retains the particles during a sufficient period, and then releases them and disrupts the cells, which thus makes it possible to make the nucleic acids accessible. The particles retained by the partition, which are made of glass, plastic or a metal such as zirconium, may have different shapes, and have a diameter of about 0.1 to 0.15 mm. One particle is assigned to breaking the partition, and has a diameter of about 1 to 4 mm, preferably 3 mm. The apparatus for subjecting to an alternating movement is a stirrer of the Biospec(copyright) trade mark.
The document EP-A-0,288,618 discloses a method of cell lysis, including microorganisms, in order to release subcellular constituents including DNA and RNA into solution. The biological sample is placed in a container with particles of different sizes. The container is then subjected to sonication until the cells release their constituents. The ultrasound causes the particles to vibrate through the sample, which causes the disruption of the cells by shearing. The particles are glass beads having a diameter of between 0.05 and 1 mm. The sonication time is 10 minutes at room temperature. The RNA and DNA are released in a manner accessible to genetic nucleic hybridization probes.
The document U.S. Pat. No. 5,464,773 discloses an apparatus for lysing cells, without destroying the subcellular constituents, in order in particular to release the RNA and DNA so as to subsequently carry out a hybridization. The container used contains two sizes of beads made of zirconium, glass or plastic and the like. In a preferred mode, the container contains 400 xcexcl of particles of zirconium of two different sizes and weights, for example 0.7 g and 0.1 mm in diameter and 0.65 g and 0.5 mm in diameter. The movement applied to the container is a vibratory-type, in particular oscillatory-type, movement.
The document U.S. Pat. No. 4,666,850 discloses an apparatus and a method for lysing a blood sample during its centrifugation. The container receiving the blood sample contains particles or beads, made of plastic or glass, which should have a size which does not prevent the sedimentation of bacteria at the bottom of the tube during centrifugation.
The document EP-A-0,341,215 discloses a method for producing heterologous proteins from genetically transformed yeasts. To evaluate the quantity of proteins, it is simply specified that to lyse the cells, mechanical shearing forces are applied by shaking the biological sample containing the yeasts with glass beads.
The document EP-A-0,284,044 discloses a method for increasing the production of proteins in yeasts. It is indicated that the cells (yeasts), for analysing the quantity of proteins, are preferably lysed by applying a vortex-type movement to the biological sample, with glass beads having a diameter of 450-500 xcexcm at a maximum speed for one minute, three times in succession.
The document U.S. Pat. No. 4,775,622 discloses a method for expressing and recovering proteins from a yeast culture.
The methods generally used for lysing a biological sample comprising at least one microorganism, specifically for releasing at least one nucleic material of interest, essentially consist according to the prior art in that:
a biological sample in a liquid medium, comprising the microorganism to be lysed, is placed in a container,
at least one particulate material which is relatively hard and substantially inert relative to the nucleic material is placed in said container,
and the mixture of biological sample and particulate material is essentially subjected to an alternating movement, of variable amplitude and/or frequency if it is a regular movement, depending on the technique or the equipment chosen for causing the movement.
These methods which are used have some disadvantages. They are not sufficiently effective, in particular the cell lysis proves to be inadequate in quantity and quality for releasing the nucleic material.
Furthermore, they do not always make it possible to lyse cells which are reputed to be resistant to lysis, in particular the cells of Gram+bacteria, for example those of Mycobacteria.
Likewise, the use of these methods often requires the addition of additional reagents such as, for example, enzymes and/or detergents.
According to the present invention, it is found that, contrary to the teaching disclosed in U.S. Pat. No. 5,643,767, a rotating movement of the vortex type was particularly suitable for lysing microorganisms and releasing the nucleic material of interest directly, provided that certain parameters for this movement are chosen, in relation in particular to the container.
The subject of the invention is therefore a method of complete, effective and simple lysing of a biological sample comprising at least one microorganism, with no reagent and/or additional operating step during the use of the method.
The first subject of the present invention is therefore a method of lysing a biological sample comprising at least one microorganism of the bacterium type, in order to release at least one nucleic material of interest belonging to said microorganism, according to which:
said biological sample in a liquid medium is placed in a container,
at least one particulate material which is relatively hard and substantially inert relative to the nucleic material is placed in said container,
the mixture of biological sample and particulate material is subjected to a movement,
characterized in that, in combination:
the movement chosen is of the vortex type, and corresponds to the following conditions:
the particulate material consists of beads having a diameter of between 90 and 150 xcexcm, and
the apparent volume of the beads, Vb, and the volume of the liquid sample, Ve, are linked by the relationship Ve=xcex1.Vb, with a between 1.4 and 10 when the container is in tubular form, and xcex1 less than or equal to 2.1 when the container is in disk form,
whereby, without addition of reagent and/or additional operating step, the nucleic material is released directly into the liquid medium in the native state and in a state accessible to any reagent.
A second subject of the invention is a method of lysing a biological sample comprising at least one microorganism of the yeast type, in order to release at least one nucleic material of interest belonging to said microorganism, according to which:
said biological sample in a liquid medium is placed in a container,
at least one particulate material which is relatively hard and substantially inert relative to the nucleic material is placed in said container,
the mixture of biological sample and particulate material is subjected to a movement, characterized in that, in combination:
the movement chosen is of the vortex type, and corresponds to the following conditions:
the particulate material consists of beads having a diameter of about 500 xcexcm, and
the apparent volume of the beads, Vb, and the volume of the liquid sample, Ve, are linked by the relationship Ve=xcex1.Vb, with a between 1.4 and 10 when the container is in tubular form, and xcex1 less than or equal to 2.1 when the container is in disk form,
whereby, without addition of reagent and/or additional operating step, the nucleic material is released into the liquid medium in the native state and in a state accessible to any reagent.
Using the method according to the invention, the nucleic material is obtained in the native state, which means that it is essentially not degraded, for example not denatured, or that it conserves practically all the characteristics or properties which it had inside the cell or organism from which it is extracted.
Moreover, the method according to the invention is sufficiently effective to allow the lysis of a single microorganism, which is of particular importance for many methods of biological analysis, especially for the techniques of amplification or analysis by hybridization of nucleic probe(s).
As regards the nucleic material, the present invention offers the decisive advantage of releasing said material in a manner directly accessible for any subsequent protocol relating to it, for example one or more amplification primers, one or more hybridization probes and the like.
Consequently, according to the present invention, the lysed biological sample obtained may be treated directly, that is to say with no intermediate operating step, or with no added reagent, according to any operating protocol in relation with the nucleic material of interest released, such as amplification, analysis, and the like, with any appropriate nucleic reagents, and the like.
In a preferred embodiment according to the first subject of the invention, the particulate material consists of beads having a diameter of about 100 xcexcm, for a microorganism of the bacterium type.
In another embodiment according to the first or second subject of the invention, the vortex-type movement satisfies, in addition, the following relationship:
Vb less than Ve less than Vc, according to which
Vc=xcex2.Ve, and
xcex2 is greater than or equal to 2.5, Vc being the useful volume of the container.
The containers which may be used according to the invention may be of different formats, for example in tubular form or in disk form.
It is preferable that the container is in tubular form, preferably with a U-shaped bottom. In this preferred embodiment, xcex2 is a number between 2.5 and 30.
In yet another embodiment according to the invention, the containers are in disk form and in tubular form with a U-shaped bottom (such as for example of the Falcons(copyright) type) or with a frustoconical 30 bottom (such as for example of the Eppendorf(copyright) type).
When the container is in disk form, xcex1 is a number less than or equal to 2.1, preferably less than or equal to 1.4 and xcex2 is a number greater than or equal to 9.3.
It is preferable that the container is a tube with a frustoconical bottom such as an Eppendorf tube, xcex1 being between 1.4 and 10, preferably between 1.4 and 3.3, and xcex2 being between 2.5 and 30, preferably between 2.5 and 15, still more preferably between 3.75 and 15.
It is also preferable that the container is a tube with a U-shaped bottom, such as a Falcon tube, xcex1 being between 1.4 and 10, preferably between 1.4 and 3.3, still preferably is equal to 3.3, and xcex2 being between 3 and 30, preferably between 12 and 30, still preferably is equal to 20.
In yet another embodiment according to the invention, the particulate material comprises other beads having a diameter greater than that of said beads. It is preferable that the container comprises up to 16, preferably 10 other beads. It is even more preferable that these other beads have a diameter of about 2 to 3 mm.
This additional characteristic of the method according to the invention makes it possible, for an equivalent efficiency, in particular to reduce or limit the time required for the lysis of the biological sample.
This additional characteristic also makes it possible to disrupt complex biological samples, such as living tissues, before the actual lysis itself.
In yet another embodiment according to the invention, an antifoaming substance may be added to the biological sample to a final concentration of 0.01 to 1% by volume, preferably of 0.5 to 1% by volume of the liquid sample, Ve.
It is also possible, according to another embodiment, to add a detergent of the anionic type to the biological sample to a final concentration of 2.5% of the volume of the liquid sample, Ve.
In a preferred embodiment according to the first subject of the invention, the time for putting through a vortex-type movement is at least equal to 10 seconds, preferably at least equal to 20 seconds, advantageously between 1 and 5 minutes.
The vortexing time is adjusted so as to obtain a percentage lysis at least greater than or equal to 20%.
It is also preferable, according to the specificities of the lysis protocol used, especially when beads having a larger diameter are added, that the time for putting through a vortex-type movement is about 2 minutes.
In a preferred embodiment according to the second subject of the invention, the time for putting through a vortex-type movement is between 8 and 20 minutes.
Given that the lysate obtained directly makes it possible to detect and/or quantify and/or amplify the nucleic material of interest, the present invention also relates to a method of treating a biological sample comprising one microorganism comprising a nucleic material of interest, said method comprising:
a) a lysis step, according to which:
said biological sample in a liquid medium is placed in a container,
at least one particulate material which is relatively hard and substantially inert relative to the nucleic material is placed in said container,
and the mixture of biological sample and particulate material is subjected to a vortex-type movement, according to which in combination:
the beads have a diameter of between 90 and 150 xcexcm for a microorganism of the bacterium type, and of about 500 xcexcm for a microorganism of the yeast type,
the apparent volume of the beads, Vb, and the volume of the liquid sample, Ve, are linked by the relationship Ve=xcex1.Vb, with xcex1 between 1.4 and 10 when the container is in tubular form, and xcex1 less than or equal to 2.1 when the container is in disk form,
b) the lysed biological sample, with or without the particulate material which served for the lysis, is subjected directly to an operating protocol relating specifically to the nucleic material of interest, for example nucleic amplification and/or detection and/or quantification.
Thus, for example after amplification, at least 1xc3x97102 cells/ml can be detected.
A third subject according to the invention is a container for a single use, for carrying out the method described above, characterized in that it comprises a load of a particulate material suited, depending on a predetermined volume of the biological sample, to the direct carrying out of the method described above.
xe2x80x9cBiological samplexe2x80x9d is understood to mean any sample, specimen or fraction of a biological material, which may be simple or complex, for example a living tissue or a body liquid or fluid. This biological sample contains at least one microorganism to be lysed, with or without prior treatment of the structure comprising said microorganism, for example destruction of the organization of the tissue.
xe2x80x9cMicroorganismxe2x80x9d is understood to mean any biological material, naturally comprising a closed membrane or envelope, one or more subcellular biological constituents of interest enclosed inside said membrane or envelope, namely nucleic constituents (DNA or RNA). By way of example, there may of course be mentioned prokaryotic microorganisms such as bacteria and archaebacteria, eukaryotic microorganisms such as plant or animal cells, yeasts and fungi, also various living tissues, and by extension viral particles.
The term xe2x80x9clysisxe2x80x9d is understood to mean any process which makes it possible to disrupt the abovementioned membrane or envelope in order to release the nucleic material of interest, in a complete or partial form.